RUI: Regulation of Diverse Bacterial ADP-Glucose Pyrophsophorylases

在各种行业中，对可生物降解和可再生碳源淀粉的使用需求越来越大。细菌糖原和植物淀粉生物合成途径中的限速酶是ADPG葡萄糖焦磷酸化酶(ADPG PPase，glgC基因产物)，它受各种变构效应分子的结合调节，取决于生物体的碳利用途径。对这个酶家族进行完整的分子比较将使我们能够进行合理的工程。ADPG PPase工程的成功将允许在转基因植物中过量生产淀粉。此外，转基因植物中淀粉合成的增加可以通过减少有机磷酸盐的反馈抑制来增加光合作用(和生物量)。本项目致力于对各种来源的细菌ADPG PPase进行动力学、物理和分子研究，这些来源包括球形红杆菌、红色红螺菌、胶囊红杆菌、沼泽红假单胞菌、耐辐射球菌和沙眼衣原体。本研究项目的具体目标包括：新的glgC基因的克隆、表达和重组酶的鉴定；以及利用定点、截断、嵌合体、随机突变和DNA改组等技术进行细菌ADPG PPase的蛋白质工程。该项目的综合方法将以比对研究、最近解决的结构、分子建模和硅配体对接为指导。重组ADPG PPase的表征将包括使用亲和毛细管电泳法测量配体的结合，以补充稳态动力学分析数据。一个较长期的目标包括将编码高活性细菌ADPG PPase的基因转化到拟南芥叶片中，以确定对淀粉积累、光合作用和生物量的影响。该项目非常适合在一个更大的跨学科生物技术项目的框架内，在生物化学、分析化学、分子生物学、生物信息学和分子建模的理论和实践方面培训以本科为主的机构的学生。一些研究实验和发现将被纳入到课堂和实验室活动中。参与的学生来自加州州立大学两个不同校区的三个实验室，以及来自高中和当地社区大学的暑期研究学生。学生在这个项目中获得的背景和经验将在学术和工业职业生涯中提供有吸引力的机会。

There is an increasing demand for the use of the biodegradable and renewable carbon source starch in a variety of industries. The rate-limiting enzyme in the bacterial glycogen and plant starch biosynthesis pathways is ADPGlucose Pyrophosphorylase (ADPG PPase, glgC gene product) which is regulated by the binding of various allosteric effector molecules depending on the carbon utilization pathway of the organism. A complete molecular comparison of this enzyme family will allow us to perform rational engineering. The successful engineering of ADPG PPase would allow for the overproduction of starch in transgenic plants. Further, increased starch synthesis in transgenic plants could increase photosynthesis (and biomass) by decreasing feedback inhibition by organic phosphates. This project is focused on kinetic, physical, and molecular studies of the uniquely regulated bacterial ADPG PPases from a variety of sources including Rhodobacter sphaeroides, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palustris, Deinococcus radiodurans, and Chlamydia trachomatis. The specific aims of this research project includeI: Cloning and expression of novel glgC genes and characterization of the recombinant enzymes; and II: Protein engineering of the bacterial ADPG PPases utilizing the techniques of site-directed, truncation, chimera, and random mutagenesis well as DNA shuffling. The comprehensive approach in this project will be guided by alignment studies, a recently solved structure, molecular modeling and in silico ligand docking. Characterization of the recombinant ADPG PPases will include the measurement of binding of ligands using affinity capillary electrophoresis to complement steady-state kinetic assay data. A longer term goal includes transforming genes coding for highly active bacterial ADPG PPases into Arabidopsis thaliana leaves to determine the effects on starch accumulation, photosynthesis, and biomass. This project is well suited to training students at a primarily undergraduate institution in the theory and practice of biochemistry, analytical chemistry, molecular biology, bioinformatics, and molecular modeling within the framework of a larger interdisciplinary biotechnology project. Some of the research experiments and findings will be incorporated into lecture and lab class activities. Participating students represent three laboratories at two different California State University campuses as well as summer research students from both high schools and local community colleges. The background and experience that is gained by students in this project will open attractive opportunities in both academic and industrial careers.